The desire to hasten the identification of potentially important polymers, drugs, catalysts, ceramic superconductors, phosphors, chemical and biological sensors, and other materials is a constant challenge that has prompted the use of combinatorial synthetic and screening strategies for synthesizing these materials and screening them for desirable properties. Combinatorial synthesis involves assembling a “library”, i.e. a very large number of chemically related compounds and mixtures, usually in the form of an array on a substrate surface. Combinatorial screening involves identifying which members of the array, if any, have the desirable property or properties. The array form facilitates the identification of a particular material on the substrate.
The synthesis of a surface-bound array of oligopeptides, short chain products of the condensation of amino acids, has been described in PCT Publication No. WO 90/15070 to M. C. Pirrung et al. entitled “Very Large Scale Immobilized Peptide Synthesis”, incorporated herein by reference. Oligopepeptides were chosen because they exhibit the types of binding specificity of their longer-chain polypeptide counterparts, such as proteins. The chemical properties and in particular, the binding properties of a protein depend almost entirely on the exposed surface amino acid residues of the polypeptide chain. These residues can form weak noncovalent bonds with other molecules. An effective binding between the protein, one example of a group of materials herein referred to as “receptors”, and the material that binds to the receptor, referred to herein as “binder”, generally requires that many weak bonds form simultaneously between the protein receptor and the binder. Binders include organic molecules, inorganic molecules, salts, metal ions, and the like. The bonds between the protein and the binder form at the “binding site” of the protein. The binding site is usually a cavity in the protein that is formed by a specific arrangement of amino acids that often belong to widely separated regions of the polypeptide chain and represent only a minor fraction of the total number of amino acids present in the chain. Binders must fit precisely into the binding site for effective binding to occur. The shape of these binding sites can differ greatly among different proteins, and even among different conformations of the same protein. Even slightly different conformations of the same protein may differ greatly in their binding abilities. For further discussion of the structure and function of proteins, see: Bruce Alberts et al., “Molecular Biology of the Cell”, 2nd edition, Garland Publishing, Inc., New York, 1989; and H. Lodish et al., “Molecular Cell Biology”, 4th edition, W. H. Freeman and Company, 2000.
After a receptor array is prepared, it is screened to determine which members have the desirable property or properties. U.S. Pat. No. 5,143,854 to M. C. Pirrung et al. entitled “Large Scale Photolithographic Solid Phase Synthesis of Polypeptides and Receptor Binding Screening Thereof”, which issued Sep. 1, 1992, hereby incorporated by reference, describes one such screening method. A polypeptide array is exposed to a ligand (an example of a binder) to determine which members of the array bind to the ligand. The ligands described are radioactive, or are “tagged”, i.e. attached via one or more chemical bonds to a chemical portion that fluoresces when exposed to non-ionizing, ultraviolet radiation. Thus, the attached portion, i.e. the tag, makes the binder visible by interrogation with ultraviolet radiation. Tagged molecules have also been used to aid in sequencing immobilized polypeptides as described, for example, in U.S. Pat. No. 5,902,723 to W. J. Dower et al. entitled “Analysis of Surface Immobilized Polymers Utilizing Microfluorescence Detection,” which issued May 11, 1999. Immobilized polypeptides are exposed to molecules labeled with fluorescent tags. The tagged molecules bind to the terminal monomer of a polypeptide, which is then cleaved and its identity determined. The process is repeated to determine the complete sequence of the polypeptide.
It is generally assumed that the attachment of a fluorescent tag to a potential binder only serves to make visible the otherwise invisible potential binder, and does not alter its binding properties. Since it is well known that even small changes to the structure of a molecule could affect its function, this assumption that a tagged binder, i.e. a “surrogate”, has the same binding affinity as the untagged binder may not be a valid one. Small structural changes that accompany even a conformational change of a receptor have been known to affect the binding affinity of the receptor. The tagged surrogates are structurally different from their untagged counterparts, and these structural differences could affect their binding affinities. Since binding affinities derived using tagged surrogates are suspect, the binding properties of receptors and binders should be evaluated using the untagged binder or receptor and not with a tagged surrogate.
Therefore, an object of the present invention is to provide an efficient, combinatorial method of evaluating the binding properties of untagged potential binders with receptors.
Another object of the present invention is to provide a combinatorial screening method for directly comparing the binding properties of receptors/binders with their tagged surrogates.
Additional objects, advantages and novel features of the invention will be set forth in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.